Lipid Profile and Apolipoprotein E Genotyping in Stroke:

A Case-Control Study

Mustafa Serteser¹, Sophie Viskikis², Tomris Ozben³, Bernard Herbeth², Sevin Balkan⁴, Gerard Siest²

¹Afyon Kocatepe University, School of Medicine, Department of Biochemistry, Inonu Bulvari,03200, Afyon, Turkey,²Centre de Medecine Preventive Upes-Interaction-Gene-Environment, Universite Henri Poincare, Nancy I, 2, Avenue du Doyen Jacques Parisot 54501, Vandoeuvre-Les-Nancy, France, Akdeniz University School of Medicine Department of Biochemistry³ and Neurology⁴, 07070, Antalya Turkey

Corresponding Author:

Mustafa SERTESER M.D.

Afyon Kocatepe University, School of Medicine, Department of Biochemistry, Inonu Bulvari,03200, Afyon, Turkey

serteser@aku.edu.tr

ABSTRACT

The possible effect of the apolipoprotein E polymorphism on the development of ischemic cerebrovascular disease has not been sufficiently investigated, and controversial results were obtained from the few existing studies. In this study, our goal was to determine the possible role of the apolipoprotein E polymorphism in stroke patients. Genotyping of apolipoprotein E carried out on 79 patients (26 thrombotic, 20 embolic, 26 lacunar, and 7 miscellaneous), and in 126 age and sex matched controls who were free of cerebrovascular disease. In addition, serum apolipoprotein E, A I, C III and B and lipoprotein (a) levels were determined. The prevalence of well-known vascular risk factors was significantly higher in the patients. The e2 allele was found to be significantly lower in patients (3.16 %) than in controls (8.34%) (c² = 4.37, p<0.05). Patients with large-vessel stroke or lacunar stroke had higher triglyceride and lower HDL levels. In all stroke subtypes, apolipoprotein A I levels were lower than those in controls, and the ratio of apolipoprotein B to A I was higher. A stepwise logistic regression showed that; the presence of vascular stroke was related to e4 allele, diabetes, high systolic blood pressure, and high apolipoprotein E serum levels, but inversely related to e2 allele and apolipoprotein A I levels. Epsilon 2 may protect individuals against stroke even if the e2 carrying patients have higher apo E levels, and e4 may be a genetic risk factor together with other well-known vascular risk factors. Large population-based studies are needed to clarify the exact relationship between stroke and lipid metabolism.

KEY WORDS:

Stroke, apo E, polymorphism

INTRODUCTION

Stroke is the third major cause of death and long term disability in industrialized countries. Intracerebral and subarachnoid haemorrhages account for only 15 % of all strokes whereas the other 85 % are caused by cerebral ischemia and can be distinguished according to the cause, clinical syndrome or the arterial distribution. Stroke due to large artery atherosclerosis causes infarcts, which are larger than 1.5 cm along with the territory of the major intracerebral arteries. Cardiac embolism also gives a similar clinical picture to large-artery atherosclerosis. In small artery occlusion, which is caused by lipohyalinosis of small perforating vessels, small brain stem or subcortical lesions of less than 1.5 cm are detected. The other rare causes of strokes are vasculitis, hemotologic disorders, migraine or oral contraceptives (van Gijn and van der Worp,1995).Cerebrovascular diseases are not only induced by classic vascular risk factors including hypertension, diabetes mellitus, cigarette smoking, but also by genetic factors (Pullicino et al.,1997).

Previously, the role of apolipoprotein E (apo E) polymorphism in atherosclerotic events has been shown (Utermann,1987). Apo E is a protein which acts as a ligand for low density lipoprotein (LDL) receptors and affects the hepatic binding, uptake and catabolism of different lipoproteins. It also has a function in the repair response to tissue injury. Increased levels of apo E concentration have been shown at sites of peripheral nerve injury and regeneration (Mahley,1988). It has been found that the gene for apo E is located on chromosome 19. Six major isoforms of the apo E gene exist, each contains a pair among three major alleles e2, e3, e4 which encode the protein isoforms E2, E3, E4 (Siest et al.,1995; Mahley,1988). Clinical and postmortem studies have shown that the e4 allele is associated with pathologies such as coronary artery diseases or Alzheimer's disease (Kosunen et al.,1995; Strittmatter and Roses, 1995, Wang et al.,1995; Corder et al.,1993;Saunders et al.,1993). In patients with ischemic cerebrovascular diseases (ICVD), only a limited number of studies clarifying the role of apolipoproteins and the role of genetic polymorphisms in lipoprotein metabolism have been performed. Studies searching for the effect of the apo E polymorphism are also rare and have produced contradictory results (Ferruci et al.,1997; Kessler et al.,1997; Schmidt et al.,1997; Kuusisto et al.,1995; Couderc et al.,1993; Pedro-Botet et al.,1992).

In this study, we aimed to find out the relationship, if one exists,between ICVD, lipids, apolipoprotein levels and apo E polymorphism. Moreover we searched for a possible relationship between apo E polymorphism and subtypes of stroke.

MATERIALS AND METHODS

Patient selection:

The study group consisted of patients admitted to the Neurology Department of Akdeniz University Hospital and to the other two major hospitals in the city, who had suffered from an ischemic event or patients admitted to the Neurology Out-patient Clinics for follow-up, who had previously been diagnosed as stroke patients within the last 15 months. Each patient underwent a complete physical and neurological examination by a neurologist. The control subjects were free of cerebrovascular diseases and were matched with the patients for sex and age on the basis of age classes ( 40-49, 50-59, 60-69, 70-80 ).

Nearly all the patients underwent computerized tomography (CT) or magnetic resonance imaging (MRI) and electrocardiography (ECG) analysis. On the basis of clinical symptoms and findings of diagnostic tools, the patients were assigned into one of the following categories: (1)- Large vessel disease-strokes due to pathologies of extracranial arteries either thrombotic or embolic, (2)- Lacunar stroke due to small deep infarctions in the territory of small perforating arteries of the brain where localization of these infarcts was confirmed by CT or MRI and the matched clinical symptoms, (3) Stroke due to miscellaneous causes which includes cardiac emboli or unknown pathomechanisms. The consciousness level (based on the Glasgow Coma Scale), functional status (based on the Barthel Index) and the level of disability (based on the Rankin Scale) were also determined in patients.

Potential confounders:

Vascular risk factors and associated vascular diseases, based on the individual's personal history, results of a physical examination and appropriate laboratory findings were recorded for patients and controls. Those included; hypertension, diabetes mellitus (DM), tobacco and alcohol consumption, use of oral contraceptives, body mass index (BMI), history of migrane, ischemic heart diseases, arrhythmias, family histories of hypertension, diabetes mellitus and stroke. Arterial hypertension was considered to be present if an individual had a history of hypertension or was using antihypertensive agents or if the systolic blood pressure (SBP) exceeded 140 mmHg or the diastolic blood pressure (DBP) exceeded 90 mmHg. DM was considered to be present if fasting glucose levels were exceeded 7.78 mmol/L or if the individual was using antidiabetic agents.

We only included patients with neurologic symptoms resulting from focal cerebral ischemia and excluded patients with intracerebral or subarachnoid haemorrhage. Patients with stroke resulting from vasculitis, migraine, oral contraceptive use or from trauma were also excluded.

Apo E genotyping:

DNA was prepared from whole blood (Miller et al.,1988). For genotyping of common apoE isoforms, amplification of apoE sequences was carried out (Hixon and Vernier,1990). Electrophoresis of the samples was performed on 10% polyacrylamid gel, after digesting the amplified products with the HhaI restriction enzyme. The detection of restriction fragments was performed by staining with ethidium bromide under UV light.

Determination of parameters of lipid metabolism:

Serum apo E concentrations were determined immunoturbidimetrically by using a kit supplied by Daicchii Pure Chemicals Co., Ltd., Tokyo, Japan. Total cholesterol, HDL-cholesterol (after precipitation procedure with phosphotungustic acid and Mg⁺² ions) and triglyceride concentrations were determined enzymatically by using CHOD/PAP and GPO/PAP methods respectively and the calculation of LDL-cholesterol was performed by using Friedewald formula. Apo AI, apo B and lipoprotein (a) [Lp(a)] levels were determined in a Behring Nephelometer. Quantification of total apo E and total apo C III particles and apo E present in the particles without apo B (apo E LP non B) and apo C III present in particles without apo B (apo C III LP non B) was carried out by an electroimmunodiffusion technique in an agarose gel supplied by Sebia (Issy-les-Moulineaux, France).

Statistical analysis:

Standard statistical procedures from the BMDP statistical software were used. For major risk factors and potential confounders, differences between case and control groups were tested by using Student's t test, ANOVA, Dunnett's test and c² analysis. As the distribution of triglyceride and Lp(a) levels were skewed, the log transformed values were used. To estimate the effect of the apo E polymorphism on the risk of stroke, whilst simultaneously adjusting for possible confounders, unconditional multiple logistic regression was used with maximum likelihood estimation of the regression coefficients and their standard errors. All the potential confounding factors were systematically tested in the regression models: sex, hypertension, diabetes, cigarette and alcohol consumption, family history of stroke and hypertension (as categorical variables), and age, SBP and DBP, BMI, cholesterol, triglyceride, apolipoprotein and lipoprotein levels (as contunious variables). The fitted model included DM, SBP and serum apo E and apo AI levels as confounders. Adjusted odds ratios for stroke were calculated for e2 allele [(e2/ e2) + (e3/ e2) + (e2/ e4)] and for e4 allele [(e3/ e4) + (e4/ e4)] by taking the e3 [(e3/ e3)] allele as a reference. Significance levels were set at 0.05 in all cases.

RESULTS

We studied 79 patients ( mean-/+SD age, 62.9-/+8.9 years, range 40-80 years, 50.6% male) and 126 controls (mean-/+SD age, 58.6+/-8.81 years, range 40-77 years, 47.6% male). The prevalence of genotypes which are shown in Table 1a. and the allele frequencies are shown in Table 1b.

Table 1a: Apo E genotypes in controls and patients

Table 1b: Apo E allelic frequency (Miscellaneous group contains patients with stroke due to cardiac embolism and with unknown cause)

 *p<0,05 Difference between controls and patients (Dunnettis test)

 No statistically significant difference was found between patients and controls for e3 and e4 allele frequencies. But e2 was found to be much more prominent in controls than in patients (c² =4.37, p<0.05). The general characteristics of the patients and controls are seen in Table 2.

Table 2: General characteristics of controls and patients.

 **p<0,05, ***p<0,001: Difference between controls and patients (Student t test or c² test)

No statistically significant sex differences were found between patients and controls but the mean age of patients was higher than those of controls (p<0.05). SBP and DBP were found to be higher in patients than in controls (p<0.001) especially in older individuals. Of the patients, 79% were found to be hypertensive and 28.4% to be diabetic. No significant difference was found between patients and controls for the family history of hypertension and DM, but on the other hand 49.5% of the patients had a positive family history for stroke, compared to 23.8% of the controls (c²=13.40, p<0.05). Current cigarette and alcohol consumption was found to be much more prominent in patients than in controls (c² =20.72, p<0.001, c² =10.02, p<0.05 respectively).

Table 3: Plasma lipid profile in controls and patients according to stroke subtypes. (Miscellaneous group contains patients with stroke due to cardiac embolism and with unknown cause)

 ¹test on log-transformed variables

*p<0.05, **p<0.01, ***p>0.001 : difference between controls and patients (Dunnettis test). 

 Table 3 shows plasma lipid profile according to stroke subtypes. Although apo E serum concentration seems to be higher in patients, no statistically significant difference was found between patients and controls, but e2 allele carrying patients had higher apo E levels than the e2 carrying controls (p<0.001) while lower apo E serum concentration was found in patients carrying e4 allele than in controls (p<0.05). Total cholesterol, triglyceride and LDL-cholesterol levels were found to be similar in patients and controls, but they were shown to be higher in e4 carrying controls than in patients (p<0.05). HDL-cholesterol levels were found to be higher in controls than in patients especially in female individuals (p<0.001). In e3 and e4 carrying controls, apo A I levels were higher than in patients (p<0.001), but we were unable to find any significant differences between patients and controls for apo B and Lp C III particles. On the other hand, total Lp E particles and Lp E NB particles were significantly higher in patients, especially in e2 and e3 carrying individuals (p<0.05).

We divided individuals according to sex ,age groups and either e2 allele carriers (e2+) or e2 allele non-carriers (e2-) in order to find out the relationship between age, sex and the occurance of stroke. We could not find any relationship between age, sex and stroke occurance in the e2+ group, but we found that, in the e2- group we had many more male patients in the 70-80 years age group than controls (c² =6.78, p<0.05). Although the number of female patients in this group was high, there was no statistical significance of this.

No statistically significant difference in cholesterol and triglyceride levels was found between stroke subtypes and controls. In thrombotic and lacunar groups, HDL-cholesterol levels were found to be lower than those in controls (p<0.05). Apo A I levels were also low in those groups (p<0.05) and the ratio of Apo B to apo A I was higher (p<0.05). Levels of Lp E and Lp E NB particles were only found to be higher in thrombotic and lacunar infarct patients (p<0.05).

The parameters showing the outcome of stroke, Glasgow Coma Scale, Barthel Index or Rankin Scale, didn't show any significant difference between male or female patients and there was also no difference according to the allele distribution and stroke subtype. But patients carrying the epsilon 2 allele seemed to show a tendency to have a better outcome than others.

Multiple logistic regression analysis indicated that apo E e4 genotype (e3 versus others) (p<0.05), DM (p<0.05), apo E level (p<0.05) and SBP (p<0.05) were significant and independent predictors of stroke, but on the other hand apo E e2 genotype and apo A I levels were found to be inversely associated with the presence of stroke (Table 4).  

Table 4:Results of the multiple logistic regression analysis.

 *e3 is the reference

**Absence of DM is reference

DISCUSSION

This study was designed as a case-control study. Due to the probable differential effect of apo E polymorphism by age, to be able to give false positive results, we had to choose the controls carefully according to the age groups designed by the cases. Therefore even though the mean ages of controls and patients differ significantly, this is attributed to the above reason and we believe that it is the best epidemiologic strategy in order to avoid confusion due to interactions between the effects of this polymorphism and age.

There are a limited number of studies performed on members of the Turkish population about apo E polymorphism. In a wide range population based study, Mahley et al. reported the allele frequencies of the Turkish population as; 6.1% for e2, 86% for e3 and 7.9% for e4 (Mahley et al.,1995). In the present study, we found 6.3% for e2, 84.6% for e3 and 9.1% for e4.Thes e results are comperable with the study of Mahley et al.

Since stroke is one of the major causes of death and long term disability, current approaches are based on preventing the occurrence of a first or recurrent stroke by reducing the risk factors, especially high blood pressure, hyperlipidemia, susceptibility to thrombus formation or platelet aggregation (Iso et al.,1989). Such measures may be partly responsible for the prevention of stroke. Besides these factors, environmental or genetic factors are also important (Graffagnino et al.,1994; Kiely et al.,1993). Recent data showed that individuals with genetic predisposition are thought to be much more prone to diseases such as coronary artery diseases or Alzheimer's disease. But data about the genetic susceptibility of stroke are insufficient and this field is of interest for researchers. 

To date, compared to other pathologies,a limited number of studies have been performed on ICVD patients in different populations. Pedro-Botet et al. reported that the e4 allele was more prevalent in ICVD patients and could be a predisposing genetic marker (Pedro-Botet et al.,1992). In our study, we were unable to find any significant difference between patients and controls for e4 allele distribution, but e4 prevalence was higher in patients with lacunar infarcts. From this point of view, our study would be compatible with that of Pedro-Botet et al., but there was no age discrimination being possibly related to the importance of e4 allele in that of Pedro-Botet et al.. Previously Kuusisto et al. reported that carrying the e4 allele cannot be regarded as an important risk factor for coronary heart disease or stroke in elderly individuals (Kuusisto et al.,1995). From this point of view; there is some conflict between our study and these other two. We found that the e4 allele is more common in ICVD patients with lacunar etiology and these findings support the previously reported data (Kessler et al.,1997). It was reported by Couderc et al. that, the E3/E2 phenotype was much more frequent in patients and concluded that the epsilon 2 gene may be a risk factor for cerebrovascular morbidity together with the associated risk factors of diabetes, hypertension and obesity (Couderc et al.,1993). In fact, in our study population the prevalence of the E3/E2 genotype was much more frequent in controls than in patients and also epsilon 2 allele frequency was found to be more common in controls. It is definitely the case that carrying an epsilon 2 allele is associated with a lower risk of ischemic stroke (Ferruci et al.,1997). These findings are consistent in young and middle-aged individuals but not confirmed in older populations (Kuusisto et al.,1995). Ferruci et al. reported that the epsilon 2 allele was associated with a lower risk of ischemic stroke but that this limited protective effect was confined to the age range 70 to 79 years (Ferruci et al.,1997). Our results are consistent with this study. We also found that the epsilon 2 allele was much more prominent in controls than in patients but we could not detect any age range in which epsilon 2 was much more protective. We observed that individuals within the age range of 70 to 80 years whoever carrying the epsilon 4 allele,were much more prone to stroke than others. This conflict may be due to the mean age of the selected subjects. In the study by Ferruci et al., the subjects were more than 70 years old and the authors concluded that results should be verified with a wider age range. In our study population, the age range was from 40 to 80 years and it can be confirmed that epsilon 2 allele is associated with a lower incidence of stroke except in the age group between 70 and 80 years. Schmidt et al. determined the apo E polymorphism in patients with microangiopathy -related cerebral damage which manifests itself with white matter abnormalities and lacunar lesions in MRI in elderly individuals (Schimidt et al.,1997). It was concluded that the E3/E2 genotype was significantly and independently associated with such abnormalities. In fact in our study of patients with lacunar infarcts, epsilon 4 was the frequently associated allele when compared with controls.It is also important to note that epsilon 2 allele carrying patients had higher apo E and Lp E non B levels. 

There are some existing results which have been published showing that the concentration of apo E is an important factor for the affinity of lipoproteins to specific apo E receptors and showing that higher apo E levels could accelerate cholesterol entrance into the cell and consequently down-regulate LDL receptors which could give increased cholesterol levels in plasma (Bohnet et al.,1996). Our results are confirmed by measuring Lp E non B particles which correspond to apo E being located in HDL-like particles where the E2 isoform is located. As was reported in previous studies we observed higher total cholesterol and triglyceride levels in individuals carrying the epsilon 4 allele (Dallongeville et al.,1992; Eto et al.,1986). We could not find any relationship in cholesterol levels between patients and controls even if epsilon 2 carrying patients had higher (but not significant) cholesterol levels than controls. These data may support the previous hypothesis that the role of hypercholesterolemia in ICVD is still questionable (Kessler et al.,1997;Prospective studies collaboration,1995). However we could hypothesize that the high concentration of apo E in epsilon 2 carrier patients could be the cause of stroke in our study. Larger studies are needed to define the exact relationship between stroke and lipid metabolism. 

McCarron et al. reported that epsilon 2 allele does not appear to be protective for ICVD, but in our study the number of controls carrying epsilon 2 allele was much more prominent than those in patients (McCarron et al.,1999). 

Apolipoproteins could be better for the discrimination of atherosclerotic events than lipids (Pedro-Botet et al.,1992). Apo A I, which activates lecithin cholesterol acyl transferase, is the main protein found in HDL-cholesterol and helps the esterification of cholesterol. Apo B is the main protein component of LDL-cholesterol and functions in the transportation of cholesterol from the liver to the vessel cells. Several studies have confirmed that determination of HDL-cholesterol or LDL-cholesterol alone did not show any prognostic power but apo A I and apo B measurements, especially the ratio of Apo B/Apo A I would be helpful in assessing the risk of atherosclerotic events. In previously reported data, no difference was found in Apo A I and Apo B levels between patients with ICVD and controls (Pedro-Botet et al.,1992; Adams et al.,1989). Woo et al. found low Apo A I levels in patients with cortical and lacunar infarcts and concluded the protective role of high Apo A I concentrations against stroke (Woo et al.,1991). In our study we could not find any statistical significance in Apo B levels between patients and controls but Apo A I levels were found to be lower in patients than in controls regardless of stroke subtypes. When we calculated the ratio of Apo B/Apo A I, it was found to be significantly higher in patients which supports the idea that the higher the ratio, the greater the risk of atherosclerotic events (Steinmetz et al.,1995;Kottke,1986; Kottke et al.,1986). High levels of Lp(a) have been found to be a major inherited risk factor associated independently with premature heart disease and stroke (Zhuang et al.,1993; Agnani et al.,1991; Woo et al.,1991). It was reported that epsilon 2 allele was found to decrease Lp(a) levels whereas epsilon 4 increases them (de Knijff et al.,1991). Although Lp(a) levels were found to be higher in patients, we couldn't find any effect of the apo E polymorphism on them. There was also no difference in Lp(a) levels according to stroke subtypes. 

In conclusion, stroke with a vascular origin could be related to the epsilon 4 allele together with the other well-known vascular risk factors.Even though the high levels of apo E were associated with the epsilon 2 carrying patients rather than than the controls, the protective effect of carrying epsilon 2 allele in the occurrence of stroke and the role of apo E should be investigated in a large population-based study to find out the exact relationship between stroke and apo E levels and polymorphism. 

ACKNOWLEDGMENTS

This work has been partly supported by FEBS short term fellowship.

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